Even though the number of genes in the human genome is much smaller than initially thought (around 20 000), the complexity and heterogeneity of human biology in health and disease has proven overwhelming. Improved knowledge and methods to define different phenotypic states are critically sought. This is driven both by basic research with the emerging Systems Biology dogma and more importantly by the realization that healthcare needs to be more personalized leading to needs to adjust/improve both drug development and disease management by development and use of biomarkers as tools for disease characterization, patient stratification and evaluation of efficacy and potential adverse effects of a treatment.
Proteins as a class have great promise as a source of biomarkers since they are the main functional actors in biological systems and define the phenotype. Not all medical indications have a direct genetic component and neither genome data nor mRNA expression data are fully predictive of protein amounts or protein states and variants.
Tissue based protein biomarkers are likely needed for detailed disease characterization (such as pathway/drug escape monitoring during cancer treatment) and patient stratification/detailed diagnosis due to potential distortion and the inherent dilution of information during leakage of proteins into circulation. In addition the applicability of “tissue based biomarkers” is increasing rapidly with the exploration of diagnostic information in liquid biopsies such as circulating tumor cells, exosomes and other micro-vesicles. Currently there are three main groups of methodologies specifically suited for general analysis of proteins directly in tissue/cells/micro-vesicles or extracted from tissue/cells/micro-vesicles:
In Immunohistochemistry (IHC), formalin-treated tissue sections are interrogated with labeled antibodies (and other stains for morphology) and read-out is performed by the use of advanced microscopes. This is potentially the most information-rich method since proteins are measured in their biological context and heterogeneity between cell types can be assessed. However, it has limited ability to detect protein isoforms, limited multiplexing capabilities and you need an expensive microscope. In addition, sample pre-treatment for antigen retrieval can be quite complex and generally need tailoring for different sets of targets.
In Western Blotting (WB), proteins from tissue are extracted and denatured before separated using i.e. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a porous membrane and interrogated with labeled antibodies (chemiluminiscence or fluorescence). Read-out is performed using either a scanner or a CCD-based system. However, WB has severe limitations for more advanced applications such as for protein biomarkers. It has limited quantification capability, limited sensitivity and dynamic range, limited abilities to distinguish isoforms and very limited multiplexing capabilities. The workflow is also relatively complicated and difficult to scale up for high throughput.
In MRM-MS/MS based methods, proteins are extracted and denatured before being digested into peptides [1]. Proteins are then quantified via their proteotypic peptides using a special form of MS read-out (MRM or SRM). However it has several important challenges: (a) low sensitivity without advanced pre-fractionation, (b) quantification variability, (c) long time to achieve results and relatively low throughput, (d) general detection of protein isoforms will likely be a challenge for many years to come.
Perhaps most important is the fact that for all of the above methodologies it will be difficult to implement standardized and automated solutions in a low cost and easy to use platform that can promote wide adoption and measurements of very large number of samples. Hence, in summary there is still a need for improved protein analytics for analyzing clinical samples of tissue, cells or micro-vesicles.
Affinity based proximity assay technology offers a novel and highly versatile toolbox for analysis of proteins and protein features. It is based on affinity binders coupled to oligonucleotides and the conditional creation of target specific nucleic acid molecules as a result of a combination of proximal binding of two or more affinity binders carrying different oligonucleotides and the action of nucleic acid processing enzymes. Different implementations such as Proximity Ligation Assays (PLA) [2], Proximity Extension Assays (PEA) [3], Rolling Circle Product PLA (RCP-PLA) [4] and Unfolding Proximity Probe assays [5] for measurements on proteins in solution have been described. Nucleic acid signals created as a result of proximal reactions can be detected and quantified in a number of ways including Q-PCR [3], digital detection of RCPs using fluorescence read-out [4], detection of RCPs using magnetic detection [6] and digital detection using third generation sequencing [7]. Since information about which set of affinity binders that has bound in proximity is present in the generated signal, the technology is uniquely suited to avoid the fundamental issue of cross-reactivity which stops any direct sandwich immunoassay implementation from enabling accurate multiplexed results in complex solution based protein samples. The increased specificity also opens up for increased sensitivity and ability for detection of specific isoforms and protein complexes in solution.
A special implementation of the technology (In-situ PLA [2]) has previously also been developed [8-9] to allow improved detection capabilities for proteins in Western Blotting workflows. Increased abilities include improved specificity, ability to detect isoforms such as phosphorylation directly using one signal and improved sensitivity.
SOMAmer affinity assay technology using a specifically designed category of nucleic acid aptamers allowing dual specificity building events to be incorporated using a single binder molecule have recently been developed by Larry Gold et al. [10]. This development has allowed a route to significantly reduce cross-reactivity issues for multiplexed assays in complex protein samples in solution while still keeping the specificity of a single analyte sandwich assay. An assay simultaneously detecting close to a thousand analytes has been reported based on this technology [10].
A major concern when analyzing proteins extracted from sources such as tissue biopsies, cells from culture, primary cells such as circulating tumor cells and micro-vesicles such as exosomes is being able to maintain the in-vivo distribution of protein amounts and modification states in the sample brought forward to analysis. Degradation or modification of proteins through altered enzymatic activity post sample acquisition is one of the challenges. Protein degradation or modification can be caused either by changes in the physiological state of the collected cells due to perturbations of the cells natural environment and/or new enzymatic activity coming into contact with targeted proteins during the extraction procedure. Another challenge is to achieve complete and reproducible extraction and solubilization of all the proteins present in the original sample. To control the above issues it is generally necessary to achieve rapid inactivation of enzymatic activity through protein denaturation coupled with the use of efficient solubilizing agents to extract proteins from the in-vivo architecture and avoid precipitation of denatured proteins. A preferred strategy for stringent sample processing is the use of a combination of heat and SDS as a solubilizing and denaturing agent. With the exception of some MS based workflows and WB, available protein analysis technologies are generally not compatible with such extraction strategies.